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1.
AIDS Rev ; 23(3): 153-163, 2021 06 03.
Article in English | MEDLINE | ID: covidwho-1579385

ABSTRACT

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious RNA coronavirus responsible for the pandemic of the coronavirus disease 2019 (COVID-19). Recent advances in virology, epidemiology, diagnosis, and clinical management of COVID-19 have contributed to the control and prevention of this disease, but re-positivity of SARS-CoV-2 in recovered COVID-19 patients has brought a new challenge for this worldwide anti-viral battle. Reverse transcription polymerase chain reaction (RT-PCR) tests of the SARS-CoV-2 pathogen is widely used in clinical diagnosis, but a positive RT-PCR result may be multifactorial, including false positive, SARS-CoV-2 RNA fragment shedding, reinfection of SARS-CoV-2, or re-activation of COVID-19. Re-infection of SARS-CoV-2 or re-activation of COVID-19 is an indicator of live viral carriers and isolation/treatment is needed, but SARS-CoV-2 RNA fragment shedding is not. SARS-CoV-2 RNA is recently reported to integrate into the host genome, but the far-reaching outcome is currently unclear. Therefore, it is critical for appropriate manipulation and prevention of COVID-19 to distinguish these causal factors of SARS-CoV-2 re-positivity. In this review article, we updated the current knowledge of SARS-CoV-2 re-positivity in discharged COVID-19 patients with a focus on re-infection and re-activation. We proposed a hypothetical flowchart for handling of the SARS-CoV-2 re-positive cases.


Subject(s)
COVID-19/pathology , RNA, Viral/analysis , Reinfection/virology , SARS-CoV-2/genetics , Virus Activation/genetics , Adaptive Immunity/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Viral/blood , COVID-19/diagnosis , Child , Child, Preschool , False Positive Reactions , Female , Humans , Infant , Male , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Young Adult
2.
Front Immunol ; 12: 779026, 2021.
Article in English | MEDLINE | ID: covidwho-1581330

ABSTRACT

A 26-year-old otherwise healthy man died of fulminant myocarditis. Nasopharyngeal specimens collected premortem tested negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Histopathological evaluation of the heart showed myocardial necrosis surrounded by cytotoxic T-cells and tissue-repair macrophages. Myocardial T-cell receptor (TCR) sequencing revealed hyper-dominant clones with highly similar sequences to TCRs that are specific for SARS-CoV-2 epitopes. SARS-CoV-2 RNA was detected in the gut, supporting a diagnosis of multisystem inflammatory syndrome in adults (MIS-A). Molecular targets of MIS-associated inflammation are not known. Our data indicate that SARS-CoV-2 antigens selected high-frequency T-cell clones that mediated fatal myocarditis.


Subject(s)
COVID-19/complications , Myocarditis/pathology , Myocarditis/virology , Systemic Inflammatory Response Syndrome/pathology , T-Lymphocytes/immunology , Adult , COVID-19/immunology , COVID-19/pathology , Humans , Male , Myocarditis/immunology , RNA, Viral/analysis , SARS-CoV-2 , Systemic Inflammatory Response Syndrome/immunology
3.
J Korean Med Sci ; 36(48): e328, 2021 Dec 13.
Article in English | MEDLINE | ID: covidwho-1572278

ABSTRACT

BACKGROUND: In the coronavirus disease 2019 (COVID-19) pandemic era, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (Flu), and respiratory syncytial virus (RSV) is important in the rapid differential diagnosis in patients with respiratory symptoms. Three multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been recently developed commercially in Korea: PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech); STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor); and Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene). We evaluated the analytical and clinical performances of these kits. METHODS: A limit of detection tests were performed and cross-reactivity analysis was executed using clinical respiratory samples. Ninety-seven SARS-CoV-2-positive, 201 SARS-CoV-2-negative, 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive nasopharyngeal swabs were tested using the three assays. The AdvanSure™ respiratory viruses rRT-PCR assay (AdvanSure; LG Life Sciences) was used as a comparator assay for RSV. RESULTS: Except in influenza B, in SARS-CoV-2 and influenza A, there were no significant differences in detecting specific genes of the viruses among the three assays. All three kits did not cross-react with common respiratory viruses. All three kits had greater than 92% positive percent agreement and negative percent agreement and ≥ 0.95 kappa value in the detection of SARS-CoV-2 and flu A/B. Allplex detected RSV more sensitively than AdvanSure. CONCLUSION: The overall performance of three multiplex rRT-PCR assays for the concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. These kits will promote prompt differential diagnosis of COVID-19, influenza, and RSV infection in the COVID-19 pandemic era.


Subject(s)
COVID-19/diagnosis , Influenza, Human/diagnosis , Multiplex Polymerase Chain Reaction/methods , Nasopharynx/virology , RNA, Viral/analysis , Respiratory Syncytial Virus Infections/diagnosis , COVID-19/virology , Cross Reactions , Humans , Influenza A virus/genetics , Influenza A virus/isolation & purification , Influenza B virus/genetics , Influenza B virus/isolation & purification , Influenza, Human/virology , Limit of Detection , Nucleocapsid Proteins/genetics , Polyproteins/genetics , RNA, Viral/metabolism , Reagent Kits, Diagnostic , Republic of Korea , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus, Human/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/genetics , Viral Proteins/genetics
4.
J Med Virol ; 94(1): 161-172, 2022 01.
Article in English | MEDLINE | ID: covidwho-1544335

ABSTRACT

Detailed information on intrahost viral evolution in SARS-CoV-2 with and without treatment is limited. Sequential viral loads and deep sequencing of SARS-CoV-2 from the upper respiratory tract of nine hospitalized children, three of whom were treated with remdesivir, revealed that remdesivir treatment suppressed viral load in one patient but not in a second infected with an identical strain without any evidence of drug resistance found. Reduced levels of subgenomic RNA during treatment of the second patient, suggest an additional effect of remdesivir on viral replication. Haplotype reconstruction uncovered persistent SARS-CoV-2 variant genotypes in four patients. These likely arose from within-host evolution, although superinfection cannot be excluded in one case. Although our dataset is small, observed sample-to-sample heterogeneity in variant frequencies across four of nine patients suggests the presence of discrete viral populations in the lung with incomplete population sampling in diagnostic swabs. Such compartmentalization could compromise the penetration of remdesivir into the lung, limiting the drugs in vivo efficacy, as has been observed in other lung infections.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/virology , Evolution, Molecular , SARS-CoV-2/genetics , Adenosine Monophosphate/therapeutic use , Adolescent , Alanine/therapeutic use , Child , Child, Preschool , Drug Resistance, Viral , Female , Haplotypes , Humans , Infant , Lung/virology , Male , Phylogeny , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Viral Load , Virus Replication/drug effects
5.
J Clin Lab Anal ; 35(11): e24002, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1525446

ABSTRACT

BACKGROUND: The coronavirus disease 2019 (COVID-19) epidemic is still spreading rapidly around the world. Recent cases with prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA detection have been successively reported, and the phenomenon of false-negative real-time polymerase chain reaction (RT-PCR) results of SARS-CoV-2 RNA or "repositive" was also described in COVID-19 patients. METHODS: We report a 69-year-old female patient with hypertension, suspected lung tumor, and previous history of total hysterectomy for hysteromyoma who presented with moderate COVID-19 symptoms and was positive for SARS-CoV-2 RNA by RT-PCR when she traveled from the USA to China. RESULTS: The patient required second and third re-hospitalizations due to "repositive" SARS-CoV-2 throat swab test results during post-charge solitary isolation and observation, and serum SARS-CoV-2-IgG decayed rapidly before disappearing on illness Day 139 when the throat swab was still positive. The virus shedding lasted for at least 146 days (the last positive throat swab test result was on illness Day 146, and the first true-negative test result was on illness Day 151) since her initial positive test. CONCLUSION: Prolonged SARS-CoV-2 RNA viral shedding is prone to occur in an immunocompromised host, wherein changes in the host immune status can lead to repeated positive SARS-CoV-2 detection. Moreover, the SARS-CoV-2-IgG may decrease rapidly and disappear before virus removal, indicating there may be certain limitations on the protective effect of the SARS-CoV-2 antibody, which deserves clinical attention.


Subject(s)
Antibodies, Viral/blood , COVID-19/virology , Immunoglobulin G/blood , SARS-CoV-2/immunology , Virus Shedding , Aged , COVID-19/immunology , Female , Humans , RNA, Viral/analysis , SARS-CoV-2/isolation & purification
6.
Front Immunol ; 12: 736529, 2021.
Article in English | MEDLINE | ID: covidwho-1515533

ABSTRACT

Various authors have hypothesized carotid body (CB) involvement in Coronavirus Disease 2019 (COVID-19), through direct invasion or indirect effects by systemic stimuli ('cytokine storm', angiotensin-converting enzyme [ACE]1/ACE2 imbalance). However, empirical evidence is limited or partial. Here, we present an integrated histopathological and virological analysis of CBs sampled at autopsy from four subjects (2 males and 2 females; age: >70 years old) who died of COVID-19. Histopathological, immunohistochemical and molecular investigation techniques were employed to characterize Severe Acute Respiratory Syndrome - Coronavirus 2 (SARS-CoV2) viral invasion and inflammatory reaction. SARS-CoV2 RNA was detected in the CBs of three cases through Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR). In these cases, positive immunostaining for Nucleocapsid and Spike protein were also demonstrated, mainly at the level of large roundish cells consistent with type I cells, confirming direct CB invasion. In these cases, T lymphocytes showed focal aggregations in the CBs, suggestive of local inflammatory reaction. Blood congestion and microthrombosis were also found in one of the positive cases. Intriguingly, microthrombosis, blood congestion and microhaemorrages were also bilaterally detected in the CBs of the negative case, supporting the possibility of COVID-19 effects on the CB even in the absence of its direct invasion. SARS-CoV-2 direct invasion of the CB is confirmed through both immunohistochemistry and RT-PCR, with likely involvement of different cell types. We also reported histopathological findings which could be ascribed to local and/or systemic actions of SARS-CoV-2 and which could potentially affect chemoreception.


Subject(s)
COVID-19 , Carotid Body , SARS-CoV-2 , Aged , Autopsy , COVID-19/pathology , COVID-19/virology , Carotid Body/pathology , Carotid Body/virology , Coronavirus Nucleocapsid Proteins/metabolism , Female , Humans , Male , Phosphoproteins/metabolism , RNA, Viral/analysis , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism
8.
Emerg Microbes Infect ; 10(1): 2199-2201, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1505680

ABSTRACT

We report pilot studies to evaluate the susceptibility of common domestic livestock (cattle, sheep, goat, alpaca, rabbit, and horse) to intranasal infection with SARS-CoV-2. None of the infected animals shed infectious virus via nasal, oral, or faecal routes, although viral RNA was detected in several animals. Further, neutralizing antibody titres were low or non-existent one month following infection. These results suggest that domestic livestock are unlikely to contribute to SARS-CoV-2 epidemiology.


Subject(s)
COVID-19/veterinary , Host Specificity , Livestock/virology , SARS-CoV-2/pathogenicity , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/virology , Camelids, New World/virology , Cattle/virology , Chlorocebus aethiops , Disease Reservoirs/virology , Goats/virology , Horses/virology , Host Specificity/immunology , Humans , Nasal Cavity/virology , RNA, Viral/analysis , Rabbits/virology , Rectum/virology , Respiratory System/virology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sheep/virology , Species Specificity , Vero Cells , Virus Shedding , Viscera/virology
9.
Microbiol Spectr ; 9(2): e0079321, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1495010

ABSTRACT

To determine the relationship between viral kinetics and severity of disease in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we investigated the viral kinetics and compared the viral loads of patients with coronavirus disease 2019 (COVID-19; the disease caused by SARS-CoV-2), stratified by symptoms and severity. We determined the viral kinetics of 100 patients diagnosed with COVID-19 at Chosun University Hospital between February 2020 and May 2021 and analyzed the differences between asymptomatic, symptomatic, and nonsurvivor patients and between patients who died and those who survived. Clinical samples, comprising respiratory specimens (sputum samples and nasopharynx and oropharynx swab samples), were obtained at different time points of hospitalization, at 1, 3 to 5, 7, 10, 14, and 30 days. SARS-CoV-2 was detected using real-time reverse transcription-PCR (RT-PCR). All three groups, asymptomatic, symptomatic, and deceased patients, had higher numbers of viral copies at symptom onset, and the asymptomatic group had lower numbers of viral copies than the symptomatic or nonsurvivor group. Viral RNA release was detected until 30 days after symptom onset. The virus cleared up earlier in asymptomatic patients than in symptomatic and nonsurvivor patients, and it cleared up earlier in mildly affected patients than in severely affected patients. The cycle threshold values tended to be significantly lower in the group receiving steroids than in the nonsteroid group, even in the low-risk group with a pneumonia severity index of less than 90. The viral loads in patients with COVID-19 were significantly different according to disease severity and steroid use. IMPORTANCE In our study, we analyzed the viral kinetics of COVID-19 patients. Our study reveals differences in viral shedding according to the severity of disease in COVID-19 patients. Viral shedding had a longer duration in severely affected patients, and the cyclic threshold values were lower in the group receiving steroids. This study is expected to be helpful in analyzing the trend of the disease course according to steroid use and severity of SARS-CoV-2 disease.


Subject(s)
COVID-19/pathology , Severity of Illness Index , Viral Load , Virus Shedding , Aged , Aged, 80 and over , Asymptomatic Infections , COVID-19/mortality , Female , Humans , Male , Middle Aged , RNA, Viral/analysis , SARS-CoV-2/isolation & purification
10.
Sci Rep ; 11(1): 21284, 2021 10 28.
Article in English | MEDLINE | ID: covidwho-1493215

ABSTRACT

We quantified the presence of SARS-CoV-2 RNA in the air of different hospital settings and the autopsy room of the largest medical centre in Sao Paulo, Brazil. Real-time reverse-transcription PCR was used to determine the presence of the envelope protein of SARS-CoV-2 and the nucleocapsid protein genes. The E-gene was detected in 5 out of 6 samples at the ICU-COVID-19 ward and in 5 out of 7 samples at the ward-COVID-19. Similarly, in the non-dedicated facilities, the E-gene was detected in 5 out of 6 samples collected in the ICU and 4 out of 7 samples in the ward. In the necropsy room, 6 out of 7 samples were positive for the E-gene. When both wards were compared, the non-COVID ward presented a significantly higher concentration of the E-gene than in the COVID-19 ward (p = 0.003). There was no significant difference in E-gene concentration between the ICU-COVID-19 and the ICU (p = 0.548). Likewise, there was no significant difference among E-gene concentrations found in the autopsy room versus the ICUs and wards (dedicated or not) (p = 0.245). Our results show the widespread presence of aerosol contamination in different hospital units.


Subject(s)
Air Microbiology , COVID-19/virology , Hospitals , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Aerosols , Autopsy , Brazil/epidemiology , COVID-19/epidemiology , COVID-19/transmission , COVID-19 Nucleic Acid Testing , Genome, Viral , Hospital Units , Humans , Intensive Care Units , Pandemics , Pathology Department, Hospital , RNA, Viral/analysis , RNA, Viral/genetics , Virion/genetics , Virion/isolation & purification
11.
Anal Bioanal Chem ; 413(29): 7195-7204, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1482198

ABSTRACT

The pandemic of the novel coronavirus disease 2019 (COVID-19) has caused severe harm to the health of people all around the world. Molecular detection of the pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), played a crucial role in the control of the disease. Reverse transcription digital PCR (RT-dPCR) has been developed and used in the detection of SARS-CoV-2 RNA as an absolute quantification method. Here, an interlaboratory assessment of quantification of SARS-CoV-2 RNA was organized by the National Institute of Metrology, China (NIMC), using in vitro transcribed RNA samples, among ten laboratories on six different dPCR platforms. Copy number concentrations of three genes of SARS-CoV-2 were measured by all participants. Consistent results were obtained with dispersion within 2.2-fold and CV% below 23% among different dPCR platforms and laboratories, and Z' scores of all the reported results being satisfactory. Possible reasons for the dispersion included PCR assays, partition volume, and reverse transcription conditions. This study demonstrated the comparability and applicability of RT-dPCR method for quantification of SARS-CoV-2 RNA and showed the capability of the participating laboratories at SARS-CoV-2 test by RT-dPCR platform.


Subject(s)
Laboratories/organization & administration , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , Humans , Limit of Detection
12.
Emerg Microbes Infect ; 10(1): 2090-2097, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1479918

ABSTRACT

Since December 2019, coronavirus disease 2019 (COVID-19) caused by SARS coronavirus 2 (SARS-CoV-2) has spread and threatens public health worldwide. The recurrence of SARS-CoV-2 RNA detection in patients after discharge from hospital signals a risk of transmission from such patients to the community and challenges the current discharge criteria of COVID-19 patients. A wide range of clinical specimens has been used to detect SARS-CoV-2. However, to date, a consensus has not been reached regarding the most appropriate specimens to use for viral RNA detection in assessing COVID-19 patients for discharge. An anal swab sample was proposed as the standard because of prolonged viral detection. In this retrospective longitudinal study of viral RNA detection in 60 confirmed COVID-19 patients, we used saliva, oropharyngeal/nasopharyngeal swab (O/N swab) and anal swab procedures from admission to discharge. The conversion times of saliva and anal swab were longer than that of O/N swab. The conversion time of hyper sensitive-CRP was the shortest and correlated with that of CT scanning and viral detection. Some patients were found to be RNA-positive in saliva while RNA-negative in anal swab while the reverse was true in some other patients, which indicated that false negatives were inevitable if only the anal swab is used for evaluating suitability for discharge. These results indicated that double-checking for viral RNA using multiple and diverse specimens was essential, and saliva could be a candidate to supplement anal swabs to reduce false-negative results and facilitate pandemic control.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Saliva/virology , Adult , Anal Canal/virology , False Negative Reactions , Female , Humans , Male , Middle Aged , Nasopharynx/virology , Oropharynx/virology , Patient Discharge , RNA, Viral/analysis , Retrospective Studies , Young Adult
13.
Microbiol Spectr ; 9(2): e0090421, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1476401

ABSTRACT

Most individuals seroconvert after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but being seronegative is observed in 1 to 9%. We aimed to investigate the risk factors associated with being seronegative following PCR-confirmed SARS-CoV-2 infection. In a prospective cohort study, we screened health care workers (HCW) in the Capital Region of Denmark for SARS-CoV-2 antibodies. We performed three rounds of screening from April to October 2020 using an enzyme-linked immunosorbent assay (ELISA) method targeting SARS-CoV-2 total antibodies. Data on all participants' PCR for SARS-CoV-2 RNA were captured from national registries. The Kaplan-Meier method and Cox proportional hazards models were applied to investigate the probability of being seronegative and the related risk factors, respectively. Of 36,583 HCW, 866 (2.4%) had a positive PCR before or during the study period. The median (interquartile range [IQR]) age of 866 HCW was 42 (31 to 53) years, and 666 (77%) were female. After a median of 132 (range, 35 to 180) days, 21 (2.4%) of 866 were seronegative. In a multivariable model, independent risk factors for being seronegative were self-reported asymptomatic or mild infection hazard ratio (HR) of 6.6 (95% confidence interval [CI], 2.6 to 17; P < 0.001) and body mass index (BMI) of ≥30, HR 3.1 (95% CI, 1.1 to 8.8; P = 0.039). Only a few (2.4%) HCW were not seropositive. Asymptomatic or mild infection as well as a BMI above 30 were associated with being seronegative. Since the presence of antibodies against SARS-CoV-2 reduces the risk of reinfection, efforts to protect HCW with risk factors for being seronegative may be needed in future COVID-19 surges. IMPORTANCE Most individuals seroconvert after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but negative serology is observed in 1 to 9%. We found that asymptomatic or mild infection as well as a BMI above 30 were associated with being seronegative. Since the presence of antibodies against SARS-CoV-2 reduces the risk of reinfection, efforts to protect HCW with risk factors for being seronegative may be needed in future COVID-19 surges.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing , Health Personnel/statistics & numerical data , SARS-CoV-2/immunology , Adult , COVID-19/immunology , COVID-19 Nucleic Acid Testing , Cohort Studies , Coronavirus Nucleocapsid Proteins/immunology , Denmark , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , Phosphoproteins/immunology , Polymerase Chain Reaction , RNA, Viral/analysis , Seroconversion , Spike Glycoprotein, Coronavirus/immunology
14.
Microbiol Spectr ; 9(2): e0084621, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1476400

ABSTRACT

Isothermal amplification-based tests have been introduced as rapid, low-cost, and simple alternatives to real-time reverse transcriptase PCR (RT-PCR) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. The clinical performance of two isothermal amplification-based tests (Atila Biosystems iAMP coronavirus disease of 2019 [COVID-19] detection test and OptiGene COVID-19 direct plus RT-loop-mediated isothermal amplification [LAMP] test) was compared with that of clinical RT-PCR assays using different sampling strategies. A total of 1,378 participants were tested across 4 study sites. Compared with standard of care RT-PCR testing, the overall sensitivity and specificity of the Atila iAMP test for detection of SARS-CoV-2 were 76.2% and 94.9%, respectively, and increased to 88.8% and 89.5%, respectively, after exclusion of an outlier study site. Sensitivity varied based on the anatomic site from which the sample was collected. Sensitivity for nasopharyngeal sampling was 65.4% (range across study sites, 52.8% to 79.8%), for midturbinate was 88.2%, for saliva was 55.1% (range across study sites, 42.9% to 77.8%), and for anterior nares was 66.7% (range across study sites, 63.6% to 76.5%). The specificity for these anatomic collection sites ranged from 96.7% to 100%. Sensitivity improved in symptomatic patients (overall, 82.7%) and those with a higher viral load (overall, 92.4% for cycle threshold [CT] of ≤25). Sensitivity and specificity of the OptiGene direct plus RT-LAMP test, which was conducted at a single study site, were 25.5% and 100%, respectively. The Atila iAMP COVID test with midturbinate sampling is a rapid, low-cost assay for detecting SARS-CoV-2, especially in symptomatic patients and those with a high viral load, and could be used to reduce the risk of SARS-CoV-2 transmission in clinical settings. Variation of performance between study sites highlights the need for site-specific clinical validation of these assays before clinical adoption. IMPORTANCE Numerous SARS-CoV-2 detection assays have been developed and introduced into the market under emergency use authorizations (EUAs). EUAs are granted primarily based on small studies of analytic sensitivity and specificity with limited clinical validations. A thorough clinical performance evaluation of SARS-CoV-2 assays is important to understand the strengths, limitations, and specific applications of these assays. In this first large-scale multicentric study, we evaluated the clinical performance and operational characteristics of two isothermal amplification-based SARS-CoV-2 tests, namely, (i) iAMP COVID-19 detection test (Atila BioSystems, USA) and (ii) COVID-19 direct plus RT-LAMP test (OptiGene Ltd., UK), compared with those of clinical RT-PCR tests using different sampling strategies (i.e., nasopharyngeal, self-sampled anterior nares, self-sampled midturbinate, and saliva). An important specific use for these isothermal amplification-based, rapid, low-cost, and easy-to-perform SARS-CoV-2 assays is to allow for a safer return to preventive clinical encounters, such as cancer screening, particularly in low- and middle-income countries that have low SARS-CoV-2 vaccination rates.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Humans , Limit of Detection , Mass Screening , Nasopharynx/virology , Point-of-Care Systems , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Specimen Handling , Viral Load
15.
Viruses ; 13(10)2021 10 14.
Article in English | MEDLINE | ID: covidwho-1470995

ABSTRACT

The gold standard for diagnosis of SARS-CoV-2 infection has been nucleic acid amplification tests (NAAT). However, rapid antigen detection kits (Ag-RDTs), may offer advantages over NAAT in mass screening, generating results in minutes, both as laboratory-based test or point-of-care (POC) use for clinicians, at a lower cost. We assessed two different POC Ag-RDTs in mass screening versus NAAT for SARS-CoV-2 in a cohort of pediatric patients admitted to the Pediatric Emergency Unit of IRCCS-Polyclinic of Sant'Orsola, Bologna (from November 2020 to April 2021). All patients were screened with nasopharyngeal swabs for the detection of SARS-CoV-2-RNA and for antigen tests. Results were obtained from 1146 patients. The COVID-19 Ag FIA kit showed a baseline sensitivity of 53.8% (CI 35.4-71.4%), baseline specificity 99.7% (CI 98.4-100%) and overall accuracy of 80% (95% CI 0.68-0.91); the AFIAS COVID-19 Ag kit, baseline sensitivity of 86.4% (CI 75.0-93.9%), baseline specificity 98.3% (CI 97.1-99.1%) and overall accuracy of 95.3% (95% CI 0.92-0.99). In both tests, some samples showed very low viral load and negative Ag-RDT. This disagreement may reflect the positive inability of Ag-RDTs of detecting antigen in late phase of infection. Among all cases with positive molecular test and negative antigen test, none showed viral loads > 106 copies/mL. Finally, we found one false Ag-RDTs negative result (low cycle thresholds; 9 × 105 copies/mL). Our results suggest that both Ag-RDTs showed good performances in detection of high viral load samples, making it a feasible and effective tool for mass screening in actively infected children.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Viral Load/methods , Antigens, Viral/analysis , Child , Child, Preschool , Female , Humans , Male , Mass Screening/methods , RNA, Viral/analysis , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity
16.
Biosensors (Basel) ; 11(10)2021 Oct 13.
Article in English | MEDLINE | ID: covidwho-1470794

ABSTRACT

Loop-mediated isothermal amplification (LAMP) has been recently studied as an alternative method for cost-effective diagnostics in the context of the current COVID-19 pandemic. Recent reports document that LAMP-based diagnostic methods have a comparable sensitivity and specificity to that of RT-qPCR. We report the use of a portable Arduino-based LAMP-based amplification system assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 min of the amplification reaction. We show that this simple system enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 µL of reaction mix. We also spiked saliva samples with SARS-CoV-2 synthetic material and corroborated that the LAMP reaction can be successfully monitored in real time using microelectrodes in saliva samples as well. These results may have profound implications for the design of real-time and portable quantitative systems for the reliable detection of viral pathogens including SARS-CoV-2.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Humans , Microelectrodes , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , Phosphoproteins/genetics , Point-of-Care Systems , RNA, Viral/analysis , RNA, Viral/metabolism , Reaction Time , SARS-CoV-2/isolation & purification , Saliva/virology
17.
Sci Rep ; 11(1): 20471, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1469980

ABSTRACT

Dual-labeled PNA probe used RT-LAMP molecular rapid assay targeting SARS-CoV-2 ORF1ab and N genes was developed, and the analytical, clinical performances for detection of SARS-CoV-2 RNA extracted from clinical nasopharyngeal swab specimens were evaluated in this study. Data showed that this assay is highly specific for SARS-CoV-2, and the absolute detection limit is 1 genomic copy per microliter of viral RNA which can be considered to be comparable to gold-standard molecular diagnostic method real-time reverse transcriptase PCR. Both clinical sensitivity and specificity against a commercial real-time RT-PCR assay were determined as identical. In conclusion, the PNA RT-LAMP assay showed high analytical and clinical accuracy which are identical to real-time RT-PCR which has been routinely used for the detection of SARS-CoV-2.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Coronavirus Nucleocapsid Proteins/genetics , Genes, Viral , Humans , Limit of Detection , Phosphoproteins/genetics , Polyproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Proteins/genetics
18.
Biosensors (Basel) ; 11(10)2021 Oct 06.
Article in English | MEDLINE | ID: covidwho-1463553

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus (SARS-CoV)-2, is rapidly spreading and severely straining the capacities of public health communities and systems around the world. Therefore, accurate, rapid, and robust diagnostic tests for COVID-19 are crucial to prevent further spread of the infection, alleviate the burden on healthcare and diagnostic facilities, and ensure timely therapeutic intervention. To date, several detection methods based on nucleic acid amplification have been developed for the rapid and accurate detection of SARS-CoV-2. Despite the myriad of advancements in the detection methods for SARS-CoV-2, rapid sample preparation methods for RNA extraction from viruses have rarely been explored. Here, we report a rapid COVID-19 molecular diagnostic system that combines a self-powered sample preparation assay and loop-mediated isothermal amplification (LAMP) based naked-eye detection method for the rapid and sensitive detection of SARS-CoV-2. The self-powered sample preparation assay with a hydrophilic polyvinylidene fluoride filter and dimethyl pimelimidate can be operated by hand, without the use of any sophisticated instrumentation, similar to the reverse transcription (RT)-LAMP-based lateral flow assay for the naked-eye detection of SARS-CoV-2. The COVID-19 molecular diagnostic system enriches the virus population, extracts and amplifies the target RNA, and detects SARS-CoV-2 within 60 min. We validated the accuracy of the system by using 23 clinical nasopharyngeal specimens. We envision that this proposed system will enable simple, facile, efficient, and inexpensive diagnosis of COVID-19 at home and the clinic as a pre-screening platform to reduce the burden on the medical staff in this pandemic era.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/genetics , Animals , COVID-19/virology , Chlorocebus aethiops , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Point-of-Care Systems , RNA, Viral/analysis , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
19.
Sci Rep ; 11(1): 19713, 2021 10 05.
Article in English | MEDLINE | ID: covidwho-1454811

ABSTRACT

The novel coronavirus disease 2019 (COVID-19) presents with non-specific clinical features. This may result in misdiagnosis or delayed diagnosis, and lead to further transmission in the community. We aimed to derive early predictors to differentiate COVID-19 from influenza and dengue. The study comprised 126 patients with COVID-19, 171 with influenza and 180 with dengue, who presented within 5 days of symptom onset. All cases were confirmed by reverse transcriptase polymerase chain reaction tests. We used logistic regression models to identify demographics, clinical characteristics and laboratory markers in classifying COVID-19 versus influenza, and COVID-19 versus dengue. The performance of each model was evaluated using receiver operating characteristic (ROC) curves. Shortness of breath was the strongest predictor in the models for differentiating between COVID-19 and influenza, followed by diarrhoea. Higher lymphocyte count was predictive of COVID-19 versus influenza and versus dengue. In the model for differentiating between COVID-19 and dengue, patients with cough and higher platelet count were at increased odds of COVID-19, while headache, joint pain, skin rash and vomiting/nausea were indicative of dengue. The cross-validated area under the ROC curve for all four models was above 0.85. Clinical features and simple laboratory markers for differentiating COVID-19 from influenza and dengue are identified in this study which can be used by primary care physicians in resource limited settings to determine if further investigations or referrals would be required.


Subject(s)
COVID-19/pathology , Dengue/pathology , Influenza, Human/pathology , Adult , Area Under Curve , COVID-19/complications , COVID-19/virology , Cohort Studies , Dengue/complications , Dengue/virology , Diagnosis, Differential , Diarrhea/etiology , Female , Fever/etiology , Humans , Influenza, Human/complications , Influenza, Human/virology , Lymphocyte Count , Male , Middle Aged , Platelet Count , RNA, Viral/analysis , RNA, Viral/metabolism , ROC Curve , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Vomiting/etiology , Young Adult
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